It has been known for some time that a variety of cancers are caused, at least in part, by mutations to certain normal genes, termed "proto-oncogenes." Proto-oncogenes are involved in regulating normal cell growth in ways that are only now beginning to be appreciated at the molecular level. The mutated proto-oncogenes, or cancer causing genes termed "oncogenes," disrupt normal cell growth which ultimately causes the death of the organism, if the cancer is not detected and treated in time.
During normal or cancer cell growth, proto-oncogenes or oncogenes, are counterbalanced by growth-regulating proteins which regulate or try to regulate the growth of normal or cancer cells, respectively. Such proteins are termed "tumor suppressor proteins." A number of such proteins are known.
A gene that encodes a tumor suppressor protein termed p53 is frequently mutated in a number of human cancers, and the inactivation of p53 is thought to be responsible for the genesis or progression of certain cancers (Nigro et al., 1989, Nature 342:705-708), including human colorectal carcinoma (Baker et al., 1989, Science 244:217-221), human lung cancer (Takahashi et al., 1989, Science 246:491-494; Iggo et al., 1990, Lancet 335:675-679), chronic myelogenous leukemia (Kelman et al., 1989, Proc. Natl. Acad. Sci. USA 86:6783-6787) and osteogenic sarcomas (Madsuda et al., 1987, Proc. Natl. Acad. Sci. USA 84:7716-7719). Tumor cells that exhibit p53 are more sensitive to radiation treatment than tumor cells that have little or no p53. Thus, knowledge of the p53 status of tumors has significant practical applications for aiding a physician in the selection of the most appropriate treatment modality.
Despite the strong experimental evidence supporting a role for p53 in tumorigenesis, there are currently only a few methods available for determining the presence of wild type or mutant p53 protein in mammalian cells. One widely used method involves time consuming DNA sequencing of the p53 gene itself. A limitation of this approach is that the presence of a normal p53 DNA sequence is not always an accurate predictor of the presence of functional p53 protein in the cells assayed since p53 function can be masked by binding of p53 protein to endogenous cellular or viral proteins (Momand, J. et al. (1992) Cell, 69:1237-1245; Oliner, J. D. et al. (1992) Nature, 358:80-83). Furthermore, this approach is both expensive to perform and time-consuming.
Another method used for determining the presence of wild type or mutant p53 involves the use of antibodies capable of distinguishing between these two forms of p53. However, this approach also has several limitations. Firstly, many of the mutations which arise in the p53 protein are point mutations and not all such mutations can be distinguished by a limited number of antibodies. Secondly, since p53 is the most commonly mutated protein identified in human cancers, the number of antibodies necessary to detect all of the different mutant forms of p53 may be quite high; this method would be impractical and costly. Thirdly, by its very nature the use of p53 antibodies, similar to the DNA sequencing method described above, is performed on cell lysates. It is not applicable to living cells.
Other methods for determining p53 in cells are shown in the following patent applications. EPA 518 650, inventor Vogelstein, B. et al., describes a method for detecting p53 in cellular extracts using DNA sequences that are specific for p53 binding.
WO 94/11533 describes determining the presence of functional p53 in cells by measuring mRNA or protein encoded by a gene termed GADD45, which is an acronym for growth-arrest and DNA-damage inducible gene.
It is important to note that all presently used p53 assays require several days to complete and cannot be performed in vivo. That is, they cannot be performed without surgically biopsing, and lysing the tumor cells.
Considering the importance that tumor suppressor proteins play in regulating cell growth, and those studies that have shown that their absence is involved in establishing the malignant phenotype, methods have been developed to replace tumor suppressors in cancer cells that lack them. The most studied method centers on the delivery of the appropriate tumor suppressor gene to cancer cells using a viral vector. Perhaps the most studied vector is adenovirus. Partly because of this work, a considerable amount of information exists regarding the genetic properties of adenovirus, and how to construct recombinant forms of the virus. See, for example, Horwitz, M. S. Adenoviridae and their Replication, In: Fields, B. N. and Knipe, D. M., eds., Fundamental Virology, 2nd ed. New York, N.Y., Raven Press, Ltd., pages 771-813 (1991); and Jolly, D. Cancer Gene Therapy, vol. 1, pages 51-64 (1994).
Mittal et al., Virus Research, vol. 28, pages 67-90 (1993) shows an adenovirus type 5-luciferase recombinant containing the firefly luciferase gene flanked by simian virus 40 (SV 40) regulatory sequences inserted into the early region 3 of the adenovirus-5 genome.
Quantin et al., Proc. Natl. Acad. Sci. vol. 89: pages 2581-2584 (1992), discloses a recombinant adenovirus containing the beta-galactosidase reporter gene under the control of muscle-specific regulatory sequences. This recombinant virus directs the expression of beta-galactosidase in myotubes in vivo.
Akrigg, A., et al., PCT/GB92/01195, discloses recombinant adenovirus for use in the detection of a trans-acting gene function in a target eukaryotic cell.
As mentioned above, the tumor suppressor protein p53 has recently been implicated as playing a critical role in causing tumor cell death, or apoptosis, induced by radiation or certain chemotherapeutic agents. See, Lowe et al., Cell, vol. 74; pages 957-967 (1993). Thus, it will be appreciated that an assay for p53 is desired that does not have the limitations of currently used assays. In particular, an assay that permits the in vivo diagnosis of the p53 status of tumor cells, or a more rapid method of assaying a tumor biopsy for p53, would greatly aid a physician in selecting the most effective method of ridding a patient of such tumors.